Neurosphere Cells Research Articles

Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans.

SUMMARYNeural stem cells (NSCs) continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR) 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs), VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

Impact of chemokines on the properties of spinal cord-derived neural progenitor cells in a rat spinal cord lesion model.

The existence of endogenous neural progenitor cells (NPCs) in the adult spinal cord (sc) provides the potential for tailored repair therapies after spinal cord injury (SCI). This study investigates the impact of inflammatory mediators on properties of NPC cultures derived from adult rats after SCI. The Infinite Horizon impactor was used to apply 200-kdyn thoracic sc lesions in adult rats. Control groups received laminectomies to equivalent sc regions. Thoracic sc segments were taken for neurosphere cell cultures. Cell proliferation was found to be significantly higher in lesion groups. Neurosphere-derived cells differentiated into neurons, oligodendroglia, and astroglia. Lesion cultures exhibited significantly higher amounts of glial fibrillary acidic protein (GFAP) mRNA (P < 0.0005) and β-III-tubulin mRNA (P < 0.05) compared with sham animals. Neurospheres from different treatment groups exhibited the same amounts of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 mRNA. C-C chemokine receptor (CCR) expression on neurospheres was examined by real-time RT-PCR. CCR1 was expressed most consistently in mRNA levels in neurospheres from both treatment groups. After cell differentiation, CCR1 mRNA amounts decreased. CCR1 was detectable by immunohistochemistry in neurospheres and differentiated cells of both groups. Application of CCL3 during differentiation cycles led to significantly higher GFAP mRNA amounts in sham animals compared with CCL3-free cultures; in contrast, CCL3 had no impact on cell differentiation in the lesion group. In conclusion, impact SCI alters differentiation tendencies and proliferation rates of adult-derived sc NPCs. Thereby, CCR1/CCL3 promotes specifically astroglial differentiation of NPCs, which provides a potential target for future neurorestorative approaches.

Omentin-1 promotes the growth of neural stem cells via activation of Akt signaling.

Omentin is a novel adipokine, which is expressed in and released from omental adipose tissue. In the present study, the effect of omentin on neural stem cells (NSCs) was investigated. NSCs are a subtype of stem cell in the nervous system, which are able to self‑renew and generate neurons and glia for repairing neural lesions. Mouse NSCs were isolated and cultured in vitro. Treatment with recombinant omentin for 3 and 5 days significantly increased the size of NSC neurospheres (P<0.01) and enhanced NSC cell viability in normal conditions. In addition, omentin protected against the decrease in cell viability induced by the pro‑inflammatory cytokine tumor necrosis factor‑α. In the NSCs, incubation of omentin for 2, 4, 6, 8 and 16 h enhanced the phosphorylation of Akt at the Thr308 site and of AS160 at the Ser318 site, peaking 6 h after treatment. Additionally, treatment with LY294002 (10 µM), a specific inhibitor of phosphatidylinositol 3‑kinase/Akt signaling, eliminated the omentin‑induced increase in neurosphere size and cell viability. Overall, the present study provided the first evidence, to the best of our knowledge, that omentin promotes the growth and survival of NSCs in vitro through activation of the Akt signaling pathway. These results may contribute to the understanding of the role of omentin in the nervous system.

CB-18 * INTERROGATION OF SUB-CLONAL GENETIC DIVERSITY OF HUMAN GBM REVEALS GENETIC HETEROGENEITY IN TUMOUR-PROPAGATING CELLS, WHICH DISPLAY VARIABLE COMPETITIVE CAPACITY FOR TUMOUR PROPAGATION IN VIVO

BACKGROUND: Glioblastoma (GB) is a lethal malignancy whose clinical intransigence has been linked to extensive intra-clonal genetic and phenotypic diversity and the common emergence of therapeutic resistance. This interpretation embodies an implicit assumption that cancer stem cells or tumour propagating cells are themselves genetically and functionally diverse. Sub-clonal evolution and cancer stem cells (CSCs) have been considered as alternative mechanisms for disease progression but it can also be argued that they are part of the same process because clonal diversity is likely to be generated and sustained by genetically distinct CSCs, which provide the units for evolutionary selection. However, this has yet to be demonstrated in GB. METHODS: We screened primary GB tumours by SNP array to identify copy number alterations (minimum of three) that could be visualised in single cells by multi-colour FISH. Interrogation of neurosphere-derived cells (from four patients) and cells derived from secondary transplants of these same cells in NOD-SCID mice allowed us to infer the clonal and phylogenetic architectures. RESULTS: In all four cases studied by multicolour FISH there was genetic heterogeneity in the neurosphere cells, with each case showing a unique, branched phylogenetic architecture. In each case, more than one sub-clone was capable of propagating tumours in secondary transplanted mice. CONCLUSIONS: Our interrogation of sub-clonal genetic diversity of human GB has revealed for the first time that tumour-propagating cells in GB are genetically heterogeneous, with variable competitive capacity for tumour propagation in vivo. The link forged between genetic diversity, clonal architecture and propagating activity in vivo may facilitate the characterisation of mutational variants responsible for disease recurrence and therapeutic resistance in patients.

DR-03 * MECHANISMS THAT CAUSE RESISTANCE TO EGFR TARGETED THERAPIES IN GLIOMA

The epidermal growth factor receptor (EGFR), or its expression, is altered in 57% of glioma by mutation, rearrangement, altered splicing and/or focal amplification, suggesting that EGFR should be a prime target for therapy. However, attempts to therapeutically target EGFR in patients with glioma have so far failed. The fundamental reasons why EGFR inhibitors are so ineffective against glioma remain largely unknown. We used three approaches to address this question. First, we derived a range of early passage neurosphere cell lines from glioma patients. These cell lines express different forms of the mutated EGFR, including the most common alterations in glioma: the autoactivating deletion mutant EGFRvIII and the extracellular domain (ECD) point mutation A289V. Through exonic sequencing and signaling pathway analysis of these cell lines, several of which are resistant to EGFR-targeted therapeutics, we showed that activation of the ras/MEK/ERK signaling pathway led to innate resistance to EGFR-targeted drugs (to both tyrosine kinase inhibitors and EGFR-specific antibodies). Likewise, engagement of neurosphere cell lines with extracellular matrix proteins such as laminin caused resistance to EGFR-targeted therapeutics. Second, we analyzed a range of EGFR ECD cysteine mutants identified in glioma patient samples. All four mutants analyzed were found on the cell surface as inactive, preformed dimers. However, all receptor mutants had enhanced activation, as measured by autophosphorylation in response to EGFR ligands. Importantly, expression of these mutants in U87MG glioma xenografts rendered the tumors more susceptible to EGFR-targeted therapeutics than xenografts expressing wild-type EGFR. Last, we show that EGFRvIII partly mediates its tumorigenicity through promiscuous activation of other receptor tyrosine kinases, such as MET, through a FAK scaffold. Co-targeting the kinase activity of FAK significantly increased the antitumor activity of EGFR-targeted therapeutics. These studies provide new insight into the mechanisms of resistance and sensitivity to EGFR-based drugs in glioma.

TM-13 * DIMINISHED PROGRESSION OF GLIOBLASTOMA WITH A SUPPLEMENTED HIGH-FAT LOW-CARBOHYDRATE DIET

Glioblastoma [GB] is the deadliest and most malignant type of brain tumor, with a median survival less than 12 months. Research into tumor metabolism and the Warburg Effect have shed new light on an idea that puts energy disparities at the forefront for new and innovative treatment options for patients. Tumor cells have an established dependence on glucose that plays a significant role in growth and survival. The human body retains secondary energy sources that can be utilized in times of starvation and glucose deprivation. By exchanging ketones (β-hydroxybutyrate) for glucose as the primary energy source in the body we can slow the progression of glioblastoma's. Conventional methods for entering ketosis, such as the ketogenic diet (KD), have been tested clinically with variable success due to high rates of side effects and low diet palatability, reducing compliance. Therefore, we have created a supplemented high-fat low-carbohydrate (sHFLC) Diet [10%C;60%F(30%MCT);30%P] that induces the two key physiological features of the KD but is more nutritionally sound and palatable. Dietary effectiveness was tested in vitro using patient derived GB neurosphere cell lines and in vivo using NOD/SCID xenografts. In culture, individually reducing glucose and increasing ketones resulted in a significant reduction in tumor cell viability that was enhanced by the combination of low glucose and elevated ketones. In vivo we demonstrate that animals fed the sHFLC exhibited a significant decline in circulating glucose (96.01mg/dL ±14.25 vs 165.1mg/dL ±20.87, p < .0001) and elevation in circulating ketones (1.798mmol ±1.121 vs .400mmol ±.115, p < .0001), slowed tumor progression (390.18mm3 ±133 vs 1020.69mm3 ±368.9, p < .0001; at 28 days post-implant) and increased survival (46.5 ±13.16 days vs 34.5 ±4.2 days, p < .0001) compared to normal fed controls. Additionally the sHFLC better maintained animal body weight as well as overall appearance when compared to the KD (28.78g ±1.812 vs 24.45g ±5.999, p < .001).

ET-40 * ONCOLYTIC VIRUS INDUCED HMGB1 SECRETION AND ITS IMPACT ON TUMOR ANGIOGENESIS

Glioblastoma multiforme (GBM) is a difficult-to-treat, and the most aggressive of primary brain tumors. Survival is less than 2 years despite aggressive therapy. Oncolytic virus (OV) therapy has shown tremendous benefits in the treatment of GBM and other tumors. This benefit is limited by host antiviral immune responses that enhance OV clearance and tumor progression. In this study, using both in-vivo and in-vitro models of GBM, we have tested the role of High mobility group box 1 (HMGB1) in OV therapy. HMGB1 is a nuclear protein that binds and regulates transcription factors. Extracellular HMGB1 functions as damage associated molecular pattern (DAMP), it mediates the inflammatory response upon binding to inflammation mediators, such as receptor of advanced glycated end product (RAGE), toll-like receptors (TLRs) 2, 4, and 9. Here, we report that; 1) HSV1716 infection induced significant secretion of HMGB1 in human glioma cell lines and glioma neurosphere cells alone or when co-cultured with microglia. 2) Measurement of HMGB1 in serum of mice with intracranial tumors revealed an increase in serum HMGB1 levels after virus treatment. 3) HSV1716 infection enhanced human brain microvascular endothelial cells (HBMEC) migration towards infected cells, which was blocked by HMGB1 blocking antibodies. 4) HMGB1 both directly enhanced HBMEC migration and induced production of VEGF by microglia cells co-cultured with glioma cells. 5) Pharmacologic inhibitor of HMGB1 (Garbexate (100uM)), and anti-HMGB1 polyclonal blocking antibody blocked HSV1716 induced HMGB1 secretion of VEGF in vitro and in vivo. Our findings suggest that OV therapy with HSV1716 activates proangiogenic pathway by induction of HMGB1, and that tumor microenvironment contributes to it proangiogenic property. Thus we conclude that OV therapy that target HMGB1 secretion and angiogenic axis may improve virotherapy of HSV1716, and other oncolytic herpes simplex virus in tumors.

3.: Microtubule dysfunction in whispering dysphonia (DYT4 dystonia)

Whispering dysphonia (DYT4 dystonia) is a dominantly inherited disorder previously described in an Australian family. The clinical phenotype is of spasmodic dysphonia associated with additional focal or generalised dystonia and a characteristic facies and body habitus. Recently, the cause of whispering dysphonia was found to be a mutation in the TUBB4 gene, which encodes for a neuronally expressed tubulin. We aimed to determine whether microtubular function was impaired as a consequence of this TUBB4 mutation. We studied olfactory neurosphere cell lines derived from three affected individuals and three controls. Tubulin end-binding protein (EB3) was fluorescently tagged, allowing us to assess microtubule dynamics using time-lapse imaging in live cells. We found that there was a significant reduction in the density of microtubules and in the speed of microtubule formation. The results suggest that the TUBB4 mutation leads to a reduction in microtubule density and a decrease in the speed of microtubule formation, supporting the notion that microtubule dysfunction can play a role in the pathophysiology of dystonia.

Abstract B13: Successful establishment of a novel neurosphere and adherent cell line pair from autopsied pediatric glioblastoma

Abstract Background: Pediatric glioblastoma (GBM) is a lethal cancer that is generally refractory to conventional treatment strategies. The scarcity of relevant pre-clinical models for this tumor has been a key limitation to the development of novel agents for the treatment of this deadly disease. A growing body of evidence supports the role of tumor progenitor cells in gliomagenesis, with CD133+ cells, a putative marker of neural stem cells and glioma progenitor cells, representing a sub-population that has been implicated in treatment resistance. In order to facilitate basic and pre-clinical studies of pediatric GBM, including high-throughput drug screening and assessment of differential responses to agents screened, we sought to establish a novel primary cell line pair of refractory pediatric GBM consisting of a traditional adherent line paired with a multicellular tumorosphere (neurosphere) line enriched for CD133+ cells. Methods: Tumor tissue was obtained from an autopsy specimen of a 10 year old pediatric patient with refractory GBM post-XRT and temozolomide and utilized to establish two cell lines: a traditional adherent cell line using serum based media and a neurosphere line grown in serum-free media supplemented with EGF and bFGF. Adherent cells were split over 60 serial passages and neurospheres split over 30 serial passages. Both cell lines were characterized by immunocytochemistry and fluorescence-activated cell sorting (FACS). Tumorigenicity was examined by the intracranial injection of 103, 104, and 105 cells in NOD/SCID mice, and the subsequent assessment of mice survival and tumor histology. Both neurospheres and adherent cells were concurrently subjected to high-throughput in vitro drug screening. Results: Neurospheres demonstrated greater staining for stem-like marker CD133+ on FACS compared with adherent cells (25% vs. 2%, respectively). Immunofluorescent staining for markers of mature glial (GFAP), neuronal (synaptophysin, NeuN), and oligodendroglial (O4) differentiation were dim for both neurospheres and the adherent cells, while immature neural markers (TuJ1) were maintained. The expression of stem cell associated markers (BMI-1, nestin, OTX2) stained slightly stronger for neurospheres. At tumor doses studied, cells from both lines formed tumors when implanted in mice, and mice injected with 103 neurospheres had significantly decreased survival compared with mice implanted with 103 adherent cells. On high-throughput drug screening, a differential response was observed for multiple targeted agents. Conclusion: The novel cell line pair, designated BPt-R0315, has been established from a refractory pediatric GBM autopsy sample and comprises a neurosphere line enriched for CD133+ cells paired with a traditional, serum-based adherent cell line. This in vitro model system provides a unique and valuable resource for further pediatric GBM research and allows parallel screening of therapeutic agents against a line enriched for CD133+ cells versus a traditional adherent cell line. As evidence accumulates that tumor progenitor or stem-like cells contribute to gliomagenesis and therapy resistance, the identification of agents effective against CD133+ enriched pediatric GBM offers a promising new strategy against this lethal disease. Citation Format: Frank Y. Lin, Mari Kogiso, Hua Mao, Yue Wang, Lin Qi, Patricia Baxter, Andrew Walter, D. Will Parsons, Xiao-Nan Li. Successful establishment of a novel neurosphere and adherent cell line pair from autopsied pediatric glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B13.

Adult Limbal Neurosphere Cells: A Potential Autologous Cell Resource for Retinal Cell Generation

The Corneal limbus is a readily accessible region at the front of the eye, separating the cornea and sclera. Neural colonies (neurospheres) can be generated from adult corneal limbus in vitro. We have previously shown that these neurospheres originate from neural crest stem/progenitor cells and that they can differentiate into functional neurons in vitro. The aim of this study was to investigate whether mouse and human limbal neurosphere cells (LNS) could differentiate towards a retinal lineage both in vivo and in vitro following exposure to a developing retinal microenvironment. In this article we show that LNS can be generated from adult mice and aged humans (up to 97 years) using a serum free culture assay. Following culture with developing mouse retinal cells, we detected retinal progenitor cell markers, mature retinal/neuronal markers and sensory cilia in the majority of mouse LNS experiments. After transplantation into the sub-retinal space of neonatal mice, mouse LNS cells expressed photoreceptor specific markers, but no incorporation into host retinal tissue was seen. Human LNS cells also expressed retinal progenitor markers at the transcription level but mature retinal markers were not observed in vitro or in vivo. This data highlights that mouse corneal limbal stromal progenitor cells can transdifferentiate towards a retinal lineage. Complete differentiation is likely to require more comprehensive regulation; however, the accessibility and plasticity of LNS makes them an attractive cell resource for future study and ultimately therapeutic application.

Abstract 4354: miR-603 targets WIF1 to promote glioma tumorigenesis via Wnt/β-catenin pathway

Abstract Glioma is the most common and deadly brain tumor. In spite of progressive treatments, which include surgery, chemotherapy and radiotherapy, patient prognosis has not improved significantly. Thus emerges the urgent need to discover novel targets that effectively control glioma. The aim of our work is to investigate the possible roles of miR-603 in glioma tumorigenesis. We first examined miR-603 expression in glioma patient samples and cell lines using RT-PCR. Then, the effect of miR-603 on glioma cells was determined by MTS assay, neurosphere formation assay, EdU assay and cell cycle analysis. Western blot, immunofluorescence staining and luciferase assay were conducted to analyze the regulation of Wnt/β-catenin signaling pathway by miR-603. Lastly, nude mice were subject to intracranial xenograft of U87 cells to determine miR-603's function on glioma oncogenesis in vivo. Herein, we report, for the first time, that miR-603 is highly expressed in glioma tissue and cell lines (U87vlll, U87, T98G, LN229, U373 and U251), suggesting its prominence in glioma tumorigenesis (p &amp;lt; 0.01). Transfection of miR-603 inhibitor into U87 and U373 glioma cells revealed a significant inhibition of cell proliferation, cell cycle progression and neurosphere formation. Conversely, overexpression of miR-603 remarkably promoted these effects. From publicly available algorithms, we identified WIF1 as a potential target of miR-603. WIF1 is known to be a Wnt/β-catenin pathway inhibitor that impedes cancer cell proliferation. As expected, western blot analysis showed that WIF1 expression was markedly decreased in U87 and U373 cells transfected with miR-603, and increased in cells transfected with miR-603-inhibitor. Moreover, we confirmed WIF1 is a bona fide target of miR-603 by 3′UTR luciferase reporter assay. The negative correlation between WIF1 and miR-603 expression was shown by Pearson correlation in four normal brain tissue samples and 43 primary glioma tissue samples. Next, we found that overexpression of miR-603 could attenuate nuclear β-catenin levels and TOPflash luciferase activity, indicating that miR-603 inhibits Wnt/β-catenin signaling pathway. In accordance with our results, WIF1 and TCF4 depletion using specific siRNA dramatically suppressed the cell proliferation, cell cycle progression and neurosphere formation induced by miR-603, thus confirming that WIF1 and Wnt/β-catenin signaling pathway are functionally involved in miR-603 regulation of glioma cancer cell growth. Furthermore, nude mice stereotactically implanted with Lentivirus-miR-603-U87 cells in the right cerebral hemisphere displayed significant inhibition of WIF1 expression, shortened survival time and increased tumor size when compared with mice implanted with Lentivirus-miR-control-U87 cells. Collectively, miR-603 regulates glioma development via target WIF1, and could potentially be used in therapeutic applications for glioma treatment. Note: This abstract was not presented at the meeting. Citation Format: Mian Guo, Guangzhi Wang, Kevork Khadarian, Yongri Zheng, Albert D. Ha, Jia Shen. miR-603 targets WIF1 to promote glioma tumorigenesis via Wnt/β-catenin pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4354. doi:10.1158/1538-7445.AM2014-4354

Aurora-A inhibition offers a novel therapy effective against intracranial glioblastoma.

Glioblastoma remains a devastating disease for which novel therapies are urgently needed. Here, we report that the Aurora-A kinase inhibitor alisertib exhibits potent efficacy against glioblastoma neurosphere tumor stem-like cells in vitro and in vivo. Many glioblastoma neurosphere cells treated with alisertib for short periods undergo apoptosis, although some regain proliferative activity upon drug removal. Extended treatment, however, results in complete and irreversible loss of tumor cell proliferation. Moreover, alisertib caused glioblastoma neurosphere cells to partially differentiate and enter senescence. These effects were also observed in glioma cells treated with the Aurora-A inhibitor TC-A2317 or anti-Aurora-A siRNA. Furthermore, alisertib extended median survival of mice bearing intracranial human glioblastoma neurosphere tumor xenografts. Alisertib exerted similar effects on glioblastoma neurosphere cells in vivo and resulted in markedly reduced activated phosphoThr288Aurora-A and increased abnormal mitoses and cellular ploidy, consistent with on-target activity. Our results offer preclinical proof-of-concept for alisertib as a new therapeutic for glioma treatment.

P01.02 * FIBROBLAST GROWTH FACTOR 4 CONTRIBUTES TO 3-DIMENSIONAL GROWTH OF HUMAN GLIOBLASTOMA

Glioblastoma growth is driven by receptor tyrosine kinase (RTK)-mediated signals. One of the RTK systems recently coming into focus are the fibroblast growth factor (FGF) high-affinity receptors (FGFR1-FGFR4) due to mutation, overexpression or translocation in several cancer types. FGF/FGFR represents a complex signal network with essential functions in embryonic development, tissue homeostasis and wound healing but also for malignant transformation and growth as well as tumor neoangiogenesis and therapy failure. Several studies have suggested a role of FGFRs in human glioblastoma whereby the information on FGFR4 is sparse. Here we investigated whether FGFR4 as compared to FGFR1 blockade impacts on glioblastoma growth in vitro and in vivo. Both in human glioblastoma cell lines (N = 8) and primary cell cultures from clinical samples (N = 26) we found a widespread expression of several FGFs (e.g. FGF1, FGF2, and FGF5) but also a significant overexpression of FGFR1 and FGFR4 in distinct subgroups as compared to non-malignant brain primo cell cultures. Regarding FGFR1 mRNA, all glioma cell models investigated expressed in addition to the FGFR1-IIIb also the mesenchymal and more oncogenic FGFR1-IIIc splice variant. Application of the FGFR inhibitors (nintedanib, ponatinib) as well as expression of dominant-negative (dn) versions of FGFR1 and FGFR4 significantly reduced in vitro cell growth and clonogenicity in the tested glioma cell models whereby dnFGFR1 tended to be more efficient than dnFGFR4. Accordingly, both dominant-negative FGFRs induced significant apoptosis whereby the effects of dnFGFR1 were again significantly stronger. Surprisingly, the inhibitory effects on anchorage-independent growth in soft agar were opposite with significant mitigation by dnFGFR1 but almost complete blockade by dnFGFR4 in the majority of the glioblastoma models analysed. Additionally, neurosphere formation, indicative for the presence of glioma stem cells, was profoundly reduced by both dnFGFRs. Interestingly, FGFR4 belonged to those genes significantly overexpressed in the cancer stem cell compartment (N = 16; mRNA expression arrays of neurosphere versus adherent cell culture). Accordingly, growth of two out of three human glioblastoma xenografts analysed in SCID mice was completely inhibited by dnFGFR4 while only retarded by dnFGFR1. Summarizing our data substantiates a significant contribution of FGF/FGFR-mediated signals to different aspects of glioblastoma aggressiveness and suggests a particular role of FGFR4 in stemness and three-dimensional in vivo growth.

P01.20 * EPIGENETIC TREATMENT AND INDUCTION OF DIFFERENTIATION IN GLIOBLASTOMA MULTIFORME NEUROSPHERE CELLS LEADS TO DOWNREGULATION OF EGFR, EGFRVIII AND NESTIN TOGETHER WITH REDUCED COLONY FORMATION IN VITRO

It is suggested that Glioblastoma Multiforme (GBM) tumors contain a subpopulation of cells with stem cell features, termed brain cancer stem-like cells (bCSC), which are thought to be involved in tumorigenesis, treatment resistance and recurrence. The epidermal growth factor receptor (EGFR) and the constitutively active deletion variant, termed EGFRvIII, are frequently expressed in GBM and EGFRvIII is correlated to chemo- and radiation resistance and poor patient prognosis. Studies suggest that EGFRvIII is linked to the bCSC subpopulation and that its expression can be regulated by epigenetic treatment. The bCSC are capable of differentiating into more mature cells, and it has been proposed differentiation therapy of bCSC could reduce the self-renewing capacity and decrease the production of clones otherwise able to populate the tumor. Here we wanted to elucidate how the EGFRvIII expression is regulated by epigenetic events during induced differentiation, and which role it plays in the tumorigenic potential. A GBM neurosphere cell culture containing bCSC and expressing endogenous EGFR and EGFRvIII under serum-free conditions was used. When inducing differentiation of the GBM neurospheres by treatment with serum this led to a downregulation of EGFR and EGFRvIII expression. However, upon transfer of the neurospheres back to NB serum-free media the EGFRvIII expression was re-gained. When treating the cells over time with a HDAC inhibitor (TSA) the expression of EGFR, EGFRvIII and the stem cell marker Nestin was decreased. Furthermore, clonal growth in vitro was reduced in a dose-dependent manner by treatment with TSA as shown by a reduction of neurosphere colonies formed in soft agar. Treatment of the neurospheres with the DNA methyl-transferase inhibitor (AZA) reduced their expression of Nestin, whereas the expression of both EGFR and EGFRvIII seemed to be unaffected. When combining serum and TSA treatment, no further downregulation of EGFR/EGFRvIII could be observed. However, GFAP, CNPase and βIII-tubulin were all downregulated as compared to treatment with serum alone, suggesting that TSA impairs serum-induced differentiation of the neurospheres. In addition, treatment with a combination of AZA and serum, for 24 hours, did not affect the expression of EGFR/EGFRvIII but led to increased expression of bIII tubulin, GFAP and CNPase, suggesting induction of neurosphere differentiation. In conclusion these results indicate that treatment with drugs that induce differentiation or modify epigenetic gene expression leads to a decrease in the bCSC subpopulation, possibly via downregulation of signaling pathways, including EGFR/EGFRvIII, involved in bCSC maintenance.

Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

STEM- AND PROGENITOR-LIKE CELL CONTRIBUTION TO MALIGNANT ASTROCYTOMA HETEROGENEITY

BACKGROUND: Stem-like tumor propagating cells (TPCs) isolated from malignant astrocytoma (MA) are reportedly therapy-resistant. In contrast, progenitor-like TPCs with loss of asymmetric cell division (Cancer Cell. 20:328-40; 2011) are sensitive to temozolomide (Cancer Cell. 18:669-82; 2010). In a pediatric MA subset, an activating BRAFT1799A mutation leads to expression of hyperactivated BRafV600E kinase, and occurs with deletion of the cell cycle inhibitor CDKN2A (Cancer Res. 70:512-9; 2010). BRAFV600E-targeted therapies, including PLX4720, retard the growth of intracranial BRAFV600E MA xenografts (Clin. Cancer Res. 17:7595-604; 2011). Studies suggest that TPCs defined by current methods are not a homogeneous population and may contain subpopulations that respond differently to treatment. The divergent effects of targeted therapies on heterogeneous TPCs have yet to be explored. METHODS: Stem-like (CD133+) and progenitor-like (NG2+) cells were isolated from human MA cell lines and murine neurosphere cells with BRafV600E expression and Ink4a/Arf (CDKN2A) deletion. CD133+ and NG2+ cells labeled with proliferation markers were investigated for heterogeneity in cell cycle dynamics and division modes. Viability assays were used to determine PLX4720 IC50s, and RT-PCR was used to analyze cell cycle and polarity regulator gene expression. Pharmacological modulators of genes altered in NG2+ and CD133+ cells were tested alongside PLX4720 for anti-tumor efficacy in intracranial BRAFV600E MA xenografts. RESULTS: In murine CD133+ cells with BRafV600E expression and CDKN2A deletion, a low proliferation frequency and high rate of asymmetric division associate with reduced sensitivity to PLX4720-induced G1 arrest. Human CD133+ but not isogenic NG2+ cells have differential mRNA expression of G2-specific cell cycle regulators. Concurrently, CD133+ cells exhibit enhanced sensitivity to blockade of polo-like-kinase1 (PLK1), a cell cycle and polarity regulator. Results from ongoing analyses of the distinct proliferative capacities, cell cycle dynamics, cell division mode and responses to targeted BRafV600E and PLK1 inhibition of CD133+ and NG2+ MA cells will be presented. CONCLUSIONS: Our studies revealed that murine malignant astrocytoma TPCs are phenotypically and functionally heterogeneous. We further discovered a TPC-subpopulation specific therapy response, where CD133+ cells from malignant astrocytoma cell lines are more resistant to BRafV600E targeted inhibition but more sensitive to G2/M checkpoint kinase inhibition than NG2+ cells. This provides a potential mechanistic basis for the differential responses of stem and progenitor-like TPCs to treatment. Understanding why current treatment strategies preferentially target only certain cell-types will allow intelligent design of chemotherapeutic strategies with greater anti-tumor effects. SECONDARY CATEGORY: n/a.

PI3K and Bcl-2 inhibition primes glioblastoma cells to apoptosis through downregulation of Mcl-1 and Phospho-BAD.

Glioblastoma multiforme (GBM) is a highly malignant human brain neoplasm with limited therapeutic options. GBMs display a deregulated apoptotic pathway with high levels of the antiapoptotic Bcl-2 family of proteins and overt activity of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Therefore, combined interference of the PI3K pathway and the Bcl-2 family of proteins is a reasonable therapeutic strategy. ABT-263 (Navitoclax), an orally available small-molecule Bcl-2 inhibitor, and GDC-0941, a PI3K inhibitor, were used to treat established glioblastoma and glioblastoma neurosphere cells, alone or in combination. Although GDC-0941 alone had a modest effect on cell viability, treatment with ABT-263 displayed a marked reduction of cell viability and induction of apoptotic cell death. Moreover, combinatorial therapy using ABT-263 and GDC-0941 showed an enhanced effect, with a further decrease in cellular viability. Furthermore, combination treatment abrogated the ability of stem cell-like glioma cells to form neurospheres. ABT-263 and GDC-0941, in combination, resulted in a consistent and significant increase of Annexin V positive cells and loss of mitochondrial membrane potential compared with either monotherapy. The combination treatment led to enhanced cleavage of both initiator and effector caspases. Mechanistically, GDC-0941 depleted pAKT (Serine 473) levels and suppressed Mcl-1 protein levels, lowering the threshold for the cytotoxic actions of ABT-263. GDC-0941 decreased Mcl-1 in a posttranslational manner and significantly decreased the half-life of Mcl-1 protein. Ectopic expression of human Mcl-1 mitigated apoptotic cell death induced by the drug combination. Furthermore, GDC-0941 modulated the phosphorylation status of BAD, thereby further enhancing ABT-263-mediated cell death. Combination therapy with ABT-263 and GDC-0941 has novel therapeutic potential by specifically targeting aberrantly active, deregulated pathways in GBM, overcoming endogenous resistance to apoptosis.

Lipid metabolism enzyme ACSVL3 supports glioblastoma stem cell maintenance and tumorigenicity.

BackgroundTargeting cell metabolism offers promising opportunities for the development of drugs to treat cancer. We previously found that the fatty acyl-CoA synthetase VL3 (ACSVL3) is elevated in malignant brain tumor tissues and involved in tumorigenesis. This study investigates the role of ACSVL3 in the maintenance of glioblastoma multiforme (GBM) stem cell self-renewal and the capacity of GBM stem cells to initiate tumor xenograft formation.MethodsWe examined ACSVL3 expression during differentiation of several GBM stem cell enriched neurosphere cultures. To study the function of ACSVL3, we performed loss-of-function by using small interfering RNAs to target ACSVL3 and examined stem cell marker expression, neurosphere formation and tumor initiation properties.ResultsACSVL3 expression levels were substantially increased in GBM stem cell enriched neurosphere cultures and decreased after differentiation of the neurospheres. Down-regulating ACSVL3 with small inhibiting RNAs decreased the expression of markers and regulators associated with stem cell self-renewal, including CD133, ALDH, Musashi-1 and Sox-2. ACSVL3 knockdown in neurosphere cells led to increased expression of differentiation markers GFAP and Tuj1. Furthermore, ACSVL3 knockdown reduced anchorage-independent neurosphere cell growth, neurosphere-forming capacity as well as self-renewal of these GBM stem cell enriched neurosphere cultures. In vivo studies revealed that ACSVL3 loss-of-function substantially inhibited the ability of neurosphere cells to propagate orthotopic tumor xenografts. A link between ACSVL3 and cancer stem cell phenotype was further established by the findings that ACSVL3 expression was regulated by receptor tyrosine kinase pathways that support GBM stem cell self-renewal and tumor initiation, including EGFR and HGF/c-Met pathways.ConclusionsOur findings indicate that the lipid metabolism enzyme ACSVL3 is involved in GBM stem cell maintenance and the tumor-initiating capacity of GBM stem cell enriched-neurospheres in animals.

Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging

Neural stem/progenitor cell (NSPC) proliferation and self-renewal, as well as insult-induced differentiation, decrease markedly with age. The molecular mechanisms responsible for these declines remain unclear. Here, we show that levels of NAD(+) and nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in mammalian NAD(+) biosynthesis, decrease with age in the hippocampus. Ablation of Nampt in adult NSPCs reduced their pool and proliferation in vivo. The decrease in the NSPC pool during aging can be rescued by enhancing hippocampal NAD(+) levels. Nampt is the main source of NSPC NAD(+) levels and required for G1/S progression of the NSPC cell cycle. Nampt is also critical in oligodendrocytic lineage fate decisions through a mechanism mediated redundantly by Sirt1 and Sirt2. Ablation of Nampt in the adult NSPCs in vivo reduced NSPC-mediated oligodendrogenesis upon insult. These phenotypes recapitulate defects in NSPCs during aging, giving rise to the possibility that Nampt-mediated NAD(+) biosynthesis is a mediator of age-associated functional declines in NSPCs.

Inhibition of Notch signaling alters the phenotype of orthotopic tumors formed from glioblastoma multiforme neurosphere cells but does not hamper intracranial tumor growth regardless of endogene Notch pathway signature

Background Brain cancer stem-like cells (bCSC) are cancer cells with neural stem cell (NSC)-like properties found in the devastating brain tumor glioblastoma multiforme (GBM). bCSC are proposed a central role in tumor initiation, progression, treatment resistance and relapse and as such present a promising target in GBM research. The Notch signaling pathway is often deregulated in GBM and we have previously characterized GBM-derived bCSC cultures based on their expression of the Notch-1 receptor and found that it could be used as predictive marker for the effect of Notch inhibition. The aim of the present project was therefore to further elucidate the significance of Notch pathway activity for the tumorigenic properties of GBM-derived bCSC.Methods Human-derived GBM xenograft cells previously established as NSC-like neurosphere cultures were used. Notch inhibition was accomplished by exposing the cells to the gamma-secretase inhibitor DAPT prior to gene expression analysis and intracranial injection into immunocompromised mice.Results By analyzing the expression of several Notch pathway components, we found that the cultures indeed displayed different Notch pathway signatures. However, when DAPT-treated neurosphere cells were injected into the brain of immunocompromised mice, no increase in survival was obtained regardless of Notch pathway signature and Notch inhibition. We did however observe a decrease in the expression of the stem cell marker Nestin, an increase in the proliferative marker Ki-67 and an increased number of abnormal vessels in tumors formed from DAPT-treated, high Notch-1 expressing cultures, when compared with the control.Conclusion Based on the presented results we propose that Notch inhibition partly induces differentiation of bCSC, and selects for a cell type that more strongly induces angiogenesis if the treatment is not sustained. However, this more differentiated cell type might prove to be more sensitive to conventional therapies.